Violin Plot of Flourescence
Figure 1: The fluorescence intensity (indicated by number of
positive partitions) for the Supermix no dUTP reaction mix is
consistently high, with most values clustering above 3 on the log scale,
indicating consistently higher positive partition counts and less
variability in fluorescence. In contrast, the RT Supermix reaction mix
exhibits a broader range of positive partition values, indicating a
lower and more variable fluorescence.
Summary Table, Mean Gene Copies per µl
Figure 2: For all pathogens except measles, mean gene copies/µl were
higher in concentrations of 105 than 103
regardless of reaction mix. For most pathogens, mean gene copies/µl were
greater in the Supermix no dUTP than the One-Step RT Supermix when
concentration was held constant.
Impact
The results of this test have informed our selection of reaction mix
in subsequent tests and assay development. In the greater scheme of
public health, multiplex assays that effectively test for pathogens in
environmental samples add novelty to the field.
Data Overview
The aim of this project is to develop and validate a digital droplet
PCR multiplex assay that sensitively and specifically tests for outbreak
pathogens in wastewater liquids and solids. To start, we had to test two
different reaction mixes, which are measured by a droplet reader. Via
Poisson statistics the software estimates the DNA/RNA gene copies / µl
of sample. We collected data via laboratory testing in October of 2024,
which can be found here.
The GitHub repository for this project can be found here.